The purpose of this grant is to investigate the mechanisms by which spinal alpha2-adrenergic stimulation produces analgesia. In the last grant cycle, we focused on a cascade of alpha2-adrenoceptor stimulation leading to acetylcholine (ACh) release and nitric oxide (NO) synthesis as being central to analgesia from spinal alpha2-adrenergic agonists and systemic opioids. Two observations in these studies provide the basis for this competing renewal application. First, alpha2-adrenergic agonists increase, and alpha2-adrenergic antagonists decrease norepinephrine (NE) release in the spinal cord, contrary to what would be classically expected by presynaptic autoinhibition. We provide preliminary data that this excitatory effect of alpha2-adrenergic agonists on NE release is due to a positive feedback circuit involving nicotinic ACh receptor stimulation and NO synthesis, including the actions of a reaction product of NO and NE, 6-nitro-norepinephrine. Second, there is a fundamental shift in spinal pharmacology following nerve injury induced allodynia, in that spinal cholinergic antagonists and NO synthase inhibitors, which have minimal effects on alpha2-adrenergic analgesia in normal animals, potently inhibit the antiallodynic effects of alpha2-adrenergic agonists. The purpose of the current application is to use a variety of in vitro and in vivo methods in rats and positron emission tomography (PET) in humans to examine 1) the regulation of spinal NE release by ACh and NO and 2) the etiology of the shift in alpha2-adrenergic mechanisms of action following nerve injury. These studies should improve our understanding of how alpha2-adrenergic agonists and systemic opioids produce analgesia to acute pain and in pathologic chronic pain states.